Volume 86, Issue 1, Pages (January 2004)

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Volume 86, Issue 1, Pages 182-190 (January 2004) Polymorphism of Ca2+ Sparks Evoked from In-Focus Ca2+ Release Units in Cardiac Myocytes  Jian-Xin Shen, ShiQiang Wang, Long-Sheng Song, Taizhen Han, Heping Cheng  Biophysical Journal  Volume 86, Issue 1, Pages 182-190 (January 2004) DOI: 10.1016/S0006-3495(04)74095-3 Copyright © 2004 The Biophysical Society Terms and Conditions

Figure 1 Fast x-y imaging of an evoked Ca2+ spark beneath a loose-seal patch. (A) Six sequential confocal images taken at 16-ms intervals during depolarization from resting potential (RP, −80mV) to RP+55mV. The dashed circle denotes the rim of the pipette. (B) Time course of local Ca2+ transient. The numbers mark the time points corresponding to the images in A. Similar results were obtained in five different patches. Biophysical Journal 2004 86, 182-190DOI: (10.1016/S0006-3495(04)74095-3) Copyright © 2004 The Biophysical Society Terms and Conditions

Figure 2 Measurement of Ca2+ sparks elicited by loose-seal patch depolarization. (A) A typical example. From top to bottom: the voltage protocol, linescan image of the evoked spark, its contour plot at ΔF/F0=0.25 and its mass center (marked by the cross), and time course of local Ca2+ transient (spatially averaged over 1.1-μm width). The mass center coordinates (X, T) were determined by X=∫∫xΔF   dx   dt/∫∫DF   dx   dt and T=∫∫tΔF   dx   dt/∫∫DF   dx   dt. (B) Space-time characteristics of a spark averaged from 11 unequivocal events. Note that the pipette rim artifact in Fig. 1 was large removed in the pseudoratio (F/F0) image. Biophysical Journal 2004 86, 182-190DOI: (10.1016/S0006-3495(04)74095-3) Copyright © 2004 The Biophysical Society Terms and Conditions

Figure 3 Detectability of in-focus Ca2+ sparks. (Left) Spark detectability determined by feeding test images through the same automated spark detection algorithm. For construction of test images, the average spark in Fig. 2 B was variably scaled and randomly embedded in a noise background produced by pixel scrambling of four event-free images. (Right) Signal-to-noise characteristics. (Top) All-point histogram of ΔF/F0 from 2100 points in 21 blank traces. (Bottom) Histogram of peak ΔF/F0 in 21 blank traces. Biophysical Journal 2004 86, 182-190DOI: (10.1016/S0006-3495(04)74095-3) Copyright © 2004 The Biophysical Society Terms and Conditions

Figure 4 Morphometrics of in-focus Ca2+ sparks. (A) Evoked Ca2+ sparks from a representative patch. From left to right, linescan images, local Ca2+ transients (spatially averaged over 1.1-μm width), and spatial profiles of the first Ca2+ sparks at peak (averaged over seven scans or 5.4ms). Note the large event-to-event variation and the crisp appearance of sparks regardless of brightness. (B) Histograms of spark amplitude (left), rise time (middle), and FWHM (right). The overlay to the amplitude histogram shows the least-square, two-component Gaussian fit of the data, which peaks at 0.67 and 1.45, respectively. Biophysical Journal 2004 86, 182-190DOI: (10.1016/S0006-3495(04)74095-3) Copyright © 2004 The Biophysical Society Terms and Conditions

Figure 5 Intrinsic variability of Ca2+ sparks from individual patches. (A) Scatter plots of amplitude (left) and mean rising rate (right) of Ca2+ sparks from four different patches. Small vertical displacements were added to avoid overlapping symbols. The mean±SD was shown below respective scatter plot. (B) Diary of mean±SD for amplitude (left) and mean rising rate (right) from individual patches (in the order of data acquisition). Seven patches that displayed three or fewer evoked events were not included. n=4–17 events in each patch. Biophysical Journal 2004 86, 182-190DOI: (10.1016/S0006-3495(04)74095-3) Copyright © 2004 The Biophysical Society Terms and Conditions

Figure 6 Interrelationship between spark parameters. (A and B) Scatter plot and linear regression of spark amplitude (A) or maximal rise rate (B) as a function of activation latency (at −25mV). (C–E) Joint histograms of spark rise time and FWHM (C), or amplitude (D), or mean rising rate (E). The brightest part represents 10 or 11 events. The histograms were plotted after interpolation by the cubic convolution method of Park and Schowengerdt (1983). Biophysical Journal 2004 86, 182-190DOI: (10.1016/S0006-3495(04)74095-3) Copyright © 2004 The Biophysical Society Terms and Conditions